这项工作是由国家卫生研究院神经科学蓝图的核心赠款（P29 NS057105，BJS）的计划项目资助NS032636（BJS）的华盛顿大学和神经系统疾病的希望中心的​​支持。

1。慢病毒载体的包装。 慢病毒载体慢病毒转移载体和磷酸钙转染法将293T细胞包装所需的其他质粒共转染的产生。我们使用此协议的10 100毫米组织培养皿。它可以放大或缩小的申请而定。在293T细胞线被维持在Dulbecco的改良老鹰培养基（DMEM培养基），高血糖（4500毫克/大号），辅以10％胎牛血清（FBS），100个单位/ ml青霉素，100微克/毫升在37℃链霉素与5％ 二氧化碳培养箱。 <ol><li>种子293T细胞10 100毫米组织培养皿中的培养基（3×10 6细胞/皿）在30-40％汇合。返回的细胞培养箱。 </li><li> 20-24 h培养后，细胞密度检查。细胞在转染时约80％汇合。 </li><li>准备一个50毫升管。添加4.4毫升TE79/10（TrisHCl 1毫米，0.1毫米EDTA，pH值7.9）减去以下的质粒DNA的总量。添加100微克慢病毒转移质粒（ 图1），58微克pMDLg / pRRE，PCMV-G的31微克，25微克PRSV-REV，600μL2M 氯化钙 。轻轻混匀。 </li><li>准备另一50毫升管。加入5毫升2个哈佛商学院（0.05 M羟乙基，0.28 M氯化钠，1.5毫米的Na 2 HPO 4，pH值7.12）。 </li><li>以10毫升吸管的DNA- 氯化钙混合物，含有2个哈佛商学院，而涡旋管滴加管。 </li><li>保持30分钟，在室温（RT）的沉淀反应。 </li><li>从孵化器中删除的培养皿。由涡旋混合沉淀反应。悬浮液加入1毫升每100毫米的菜含有细胞。必须暂停增加缓慢，滴加，同时轻轻摇动菜中等。这些菜的孵化器和5小时离开。 </li><li>从文化的媒介。加入6毫升的新鲜培养基中含有6毫米钠BUtyrate到每一道菜。返回文化的孵化器。过夜培养后，如果有记者在该构造的荧光，荧光显微镜下检查报告基因的表达。通常情况下，超过80％的细胞表达报告基因，如果它是由一个无处不在的启动子（如CMV启动子）驱动。 </li><li>转染后两天（40-44小时），收集上清到2 50毫升管（每管约30毫升）从10道菜。冻结在-80°C冰箱上清液，或进入下一步骤。 </li></ol> 2。浓缩和提纯的载体<ol><li>离心收集新鲜或解冻900克（约2000转）上清为10 min，以除去上清液中的任何细胞碎片。 </li><li>将60毫升的注射器SFCA注射器到0.2微米的过滤器。 50毫升管中的上清液转移到注射器。成polyallomer离心管过滤上清液。 </li><li>以5毫升20％的蔗糖（在PBS配制），在5毫升吸管。插入吸管底部的离心管含有上清。载体上清下慢慢加入蔗糖溶液。从另一个管重复这些步骤上清。 </li><li> 11000 rpm和4°C的4小时贝克曼SW28摆动转子离心上清。 </li><li>去除上清。每个离心管中加入150微升4％的乳糖（在PBS配制）。悬浮颗粒。 </li><li>所有离心管1.5 ml管转移集中的载体。离开冰管15分钟。 </li><li>吹打混合载体悬浮。在全速（约16000克），1分钟旋转离心。 </li><li>将上清转移到一个新的1.5 ml管。最终样本分为20μL等份，并储存在-80°C冰箱。 </li></ol> 3。滴定法向量<ol><li>种子5×10 4/1毫升含10％胎牛血清的DMEM培养液培养基中12井板在HT1080细胞。 </li><li>后overnigHT文化，指望从一个井细胞和取得的细胞数量。 </li><li>请5倍系列稀释（1:5，1：25;和1:625; 1:125）与培养基集中的载体。加入我微升每个稀释载体单独井。可以复制的样本，以提高准确性。 </li><li>在每口井含有载体，没有载体以及添加1μL4毫克/毫升凝聚胺（Hexadimethrine溴化物）。混合轻轻晃动板。返回孵化器为48小时。 </li><li>从细胞培养井介质。洗PBS每口井。加入250μL1X细胞胰蛋白酶-EDTA溶液。当细胞被分离（3-5分钟），加入1毫升的培养基。吹打重悬细胞。将细胞悬液1.5毫升离心管。 </li><li>在900Ğ离心6分钟。为荧光报告基因的载体（如GFP）的，则转到步骤3.7流式细胞仪分析。对于没有记者的载体，加强实时定量PCR 3.8。 </li><li>对于1荧光载体rescent记者基因，去除上清和300微升3.7％，甲醛的PBS重悬沉淀。流式细胞仪分析，确定记者阳性细胞百分比。将代表滴度为每毫升浓缩载体转导单位（TU /毫升）。 </li></ol><img alt="式（1）" src="/files/ftp_upload/4031/4031eq1.jpg" />例如，如果1×10 5细胞与1/25μL（微升0.04）向量和30％的细胞转导记者阳性，滴度会是： <img alt="公式（2）" src="/files/ftp_upload/4031/4031eq2.jpg" />只能用稀释的阳性细胞百分比和载体的添加量计算滴度之间的线性关系下降。最终滴度应该是一个至少2不同数额的载体转导获得的平均滴度。 <ol start="8"><li>对于没有向量荧光报告基因，基因组DNA提取HT1080细胞采用QIAamp DNA Mini试剂盒（QIAGEN）根据制造商的协议。放大载体在基因组DNA序列，用引物（HIV-1 PBS / PSI地区17）5&#39;-CCGTTGTCAGGCAACGTG-3&#39;和5&#39;-AGCTGACAGGTGGTGGCAAT 3 ABI PRISM 7000序列检测系统（Applied Biosystems公司），TaqMan探针5 &#39;-FAM-AGCTCTCTCGACGCAGGACTCGGC：TAMRA-3&#39;。白蛋白基因，是基因组中单拷贝基因（2拷贝/细胞）也扩增引物5&#39;-TGAAACATACGTTCCCAAAGAGTTT-3&#39;5&#39;-CTCTCCTTCTCAGAAAGTGTGCATAT的3&#39;，探针5&#39;-FAM-TGCTGAAACATTCACCTTCCATGCAGA：TAMRA-3 “作为内部控制。确定在96孔板载体的副本数和白蛋白的PCR按以下程序的制造的指令：50℃2分钟，95°Ç10分钟，和35 95周期°Ç15秒和60°C为2分钟。十倍连续稀释已知浓度的质粒（副本代表数），其中包含的模板序列，也应放大到创建一个未知样品的定量标准曲线。将滴度为每毫升浓缩载体（国际单位/毫升）的集成单位代表。 </li></ol><img alt="公式（3）" src="/files/ftp_upload/4031/4031eq3.jpg" /> 4。转导的皮层文化鼠标使用一个两步电镀过程，如先前所述18皮质含有皮层神经元和神经胶质细胞的培养准备。在妊娠14-16天从小鼠胚胎中获得的neocortices到先前建立的胶质细胞在MEM 10％胎牛血清，20毫米的葡萄糖和24孔培养板2mM谷氨酰胺的补充单层镀。 <ol><li> 在体外经过5天，添加10μM的，胞嘧啶neoc阿糖胞苷（阿糖胞苷）ortical文化，以抑制非神经细胞的分裂。继续培养2天的细胞。 </li><li>温暖的培养基在37℃的水浴5-10分钟。更换阿糖胞苷含新鲜培养液（500μL/孔）介质。 </li><li>添加所需的教学语言（多重感染的靶细胞的数量比矢量粒子的数目）的文化载体。继续培养24 h。我们使用1-10 MOI（通常为5）在初级皮层文化。 </li><li>更换新鲜培养基培养基。继续培养。如果有记者在矢量结构基因，检查细胞，荧光显微镜后第2天转下。报告基因的表达将是可见的神经元2-7转后，取决于载体的设计和使用剂量。 </li></ol> 5。代表结果与本协议范围内产生的慢病毒载体的滴度10 8 -10 10国际单位/毫升，which是适合的多种类型的细胞在体外和体内的中枢神经系统传导。 表1和图2展示了具有代表性的结果，使用该协议所产生的载体。我们转 小鼠皮层文化表达慢病毒载体的绿色荧光蛋白（GFP）由突触素（SYN）的发起人或胶质纤维酸性蛋白（GFAP）启动子控制。七天后转，我们进行标签神经元和神经胶质细胞免疫与抗NeuN染色和抗GFAP抗体，分别。 表1和图所示。 2A后，转导与账面突触启动，超过90％的神经元（NeuN染色+细胞）的向量表达GFP并没有星形胶质细胞GFAP（+细胞）记者基因表达。当GFAP启动子用于向量构造（ 图2B），约80％的星形胶质细胞GFAP（+细胞）表达小号绿色荧光蛋白;所有GFP +细胞是共存与GFAP和GFP表达NeuN染色标记的细胞在没有证实的星形胶质细胞。这些结果表明，慢病毒载体是非常有效的提供从中枢神经系统的细胞和细胞特异性基因表达的转基因时，使用适当的发起人是可以实现的。 <img alt="图1" src="/files/ftp_upload/4031/4031fig1.jpg" /> 图1。艾滋病毒的慢病毒载体和包装质粒示意图表示。HIV-1病毒DNA显示在顶部。分为四个不同的质粒为载体生产的元素。慢病毒转移质粒包含混合5&#39;LTR的U3区的巨细胞病毒（CMV）启动子，包装信号（ψ），RRE的序列，中央polypurine的道（cPPT）的，感兴趣的基因（如更换荧光记者）随着选择的发起人，和3&#39;的LTR在其中的顺式调控序列是完全删除从U3的地区。 pMDLg，/ pRRE包含HIV-1的GAG和POL基因和CMV启动子控制下的RRE的序列。 PRSV-REV包含编码序列，由RSV启动子驱动的牧师。 PCMV-G包含CMV启动子的控制下，VSV-G蛋白基因。 PA表示，从人β-珠蛋白基因的加尾信号。 <img alt="图2" src="/files/ftp_upload/4031/4031fig2.jpg" /> 图2。记者在小鼠皮层混合培养基因表达的转携带的细胞类型特异性启动子的慢病毒载体的文化与LV - SYN-GFP（一）或（b）在5 MOI的LV-GFAP的GFP载体转。七天后转导，细胞免疫组化染色与抗NeuN染色或抗GFAP抗体。上游面板显示GFP荧光，中间面板显示免疫组化和较低的面板合并图像（GFP：绿色; NeuN染色或GFAP：红色）。 </P&gt; <table border="1"><tbody><tr><td> 向量 </td><td> 绿色荧光蛋白+细胞在神经元 </td><td> 在星形胶质细胞的GFP + </td></tr><tr><td> LV - SYN-GFP </td><td> 92.2±7.3 </td><td> 0 </td></tr><tr><td> LV-GFAP的，GFP </td><td> 0 </td><td> 78.3±11.5 </td></tr></tbody></table> 表1比较在小鼠皮层文化的表达GFP的转用慢病毒载体携带不同的促销员。 小鼠皮层文化（5×10 5/24孔板）与LV - SYN-GFP或LV-GFAP的绿色荧光蛋白转导MOI 5。七天后转固定，文化和NeuN染色或GFAP免疫组化染色。 GFP和NeuN染色/ GFAP表达细胞的数量被计算在实验条件下10个领域的图像。值代表神经元的比例（NeuN染色+细胞）星形胶质细胞GFAP（+细胞），也表达了GFP报告基因。显示的值是平均值±SD由三个独立的实验。

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Gene Ther. 22, 1511-1523 (2011).</li><li>Segura, M. M., Garnier, A., Durocher, Y., Ansorge, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+protocol+for+lentiviral+vector+mass+production.">New protocol for lentiviral vector mass production.</a> Methods Mol. Biol. 614, 39-52 (2010).</li><li>Kutner, R. H., Puthli, S., Marino, M. P., Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+production+and+concentration+of+HIV-1-based+lentiviral+vectors+using+HYPERFlask+vessels+and+anion+exchange+membrane+chromatography.">Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography.</a> BMC Biotechnol. 9, 10 (2009).</li><li>Laughlin, M. A., Chang, G. Y., Oakes, J. W., Gonzalez-Scarano, F., Pomerantz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+butyrate+stimulation+of+HIV-1+gene+expression:+a+novel+mechanism+of+induction+independent+of+NF-kappa+B.">Sodium butyrate stimulation of HIV-1 gene expression: a novel mechanism of induction independent of NF-kappa B.</a> J Acquir Immune Defic Syndr Hum Retrovirol. 9, 332-339 (1995).</li><li>Gasmi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+efficient+production+and+transduction+of+human+immunodeficiency+virus+type+1-based+vectors.">Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors.</a> J. Virol. 73, 1828-1834 (1999).</li><li>Palsson, B., Andreadis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physico-chemical+factors+that+govern+retrovirus-mediated+gene+transfer.">The physico-chemical factors that govern retrovirus-mediated gene transfer.</a> Exp. Hematol. 25, 94-102 (1997).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Lee, J. K., Chung, J., McAlpine, F. E., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-Protein+Signaling-10+Negatively+Regulates+NF-{kappa}B+in+Microglia+and+Neuroprotects+Dopaminergic+Neurons+in+Hemiparkinsonian+Rats.">Regulator of G-Protein Signaling-10 Negatively Regulates NF-{kappa}B in Microglia and Neuroprotects Dopaminergic Neurons in Hemiparkinsonian Rats.</a> J. Neurosci. 31, 11879-11888 (2011).</li><li>Shevtsova, Z., Malik, J. M., Michel, U., Bahr, M., Kugler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoters+and+serotypes:+targeting+of+adeno-associated+virus+vectors+for+gene+transfer+in+the+rat+central+nervous+system+in+vitro+and+in+vivo.">Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.</a> Exp. Physiol. 90, 53-59 (2005).</li></ol>

没有利益冲突的声明。

在这个协议中，我们已经证明，生产慢病毒载体和应用皮层文化载体。我们证明了这些方法所产生的向量与效率和细胞类型特异性的转导。当突触启动，GFP的表达是严格神经元特异性。当使用GFAP启动子，GFP的表达完全是在星形胶质细胞。如果没有细胞类型特异性表达是必需的，一个无处不在的启动也可使用。我们发现两个ubiqutin和磷酸激酶（PGK）发起人可以驱动6皮层文化高水平的基因表?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM培养液</td><td> Sigma-Aldrich公司</td><td> D5796 </td></tr><tr><td>的MEM </td><td> Invitrogen公司</td><td> 11090-081 </td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> SV3001403 </td></tr><tr><td> PBS </td><td>敏达</td><td> 21-040-CM </td></tr><tr><td>胰蛋白酶EDTA </td><td> Sigma-Aldrich公司</td><td> T3924 </td></tr><tr><td>丁酸钠</td><td> Sigma-Aldrich公司</td><td> B5887 </td></tr><tr><td> hexadimethrine溴（凝聚胺） </td><td> Sigma-Aldrich公司</td><td> H9268 </td></tr><tr><td> 293T细胞</td><td> ATCC </td><td> CRL-11268 </td></tr><tr><td> HT1080细胞</td><td> ATCC </td><td> CCL-121 </td></tr><tr><td>猎鹰100×20毫米组织的文化性E盘</td><td> BD公司</td><td> 353003 </td></tr><tr><td> 1×3半，在polyallomoer离心管中</td><td>贝克曼 - 库尔特</td><td> 326823 </td></tr><tr><td> 0.2微米注射器过滤器</td><td>康宁</td><td> 431219 </td></tr><tr><td>采用QIAamp DNA的迷你包</td><td> Qiagen公司</td><td> 51304 </td></tr></tbody></table>

production of lentiviral vectors for transducing cells from the central nervous system

在中枢神经系统（CNS）的高效基因传递是非常重要的研究基因功能，神经系统疾病的建模和开发治疗方法。慢病毒载体在中枢神经系统的神经元和其他类型的细胞转导分裂和非分裂细胞，支持转基因的持续表达，因为他们转导有吸引力的工具，并有比较大的包装能力和毒性低1-3。慢病毒载体已成功地使用在4-6 体外转导在许多神经细胞类型，并在动物7-10。 已作出很大努力，发展与改善生物安全和基因传递效率慢病毒载体。 图1描述了目前的第三代（SIN）的复制缺陷和自我失活慢病毒载体。分裂成四个质粒载体包装所需的要素。在慢病毒转FER质粒，在5&#39;长末端重复序列（LTR）的U3区与另一种病毒的强启动子取代。这一修改，允许艾滋病毒艾滋病毒基因表达11，通常需要1 Tat蛋白的独立的向量序列的转录。包装信号（Ψ）包壳和牧师响应元素（RRE的）是必不可少的需要生产高滴度的载体。中央polypurine道（cPPT）是非常重要的载体DNA，核进口转导非分裂细胞12所必需的功能。在3&#39;LTR的顺式调控序列被完全删除从U3的地区。删除复制后5&#39;LTR的反转录，从而导致转录失活两公升。的质粒pMDLg / pRRE包含HIV-1基因，GAG / POL提供结构蛋白和逆转录酶。 PRSV-REV编码冯结合RRE的高效RNA从细胞核出口。 PCMV-Ğ编码水泡性口炎病毒糖蛋白（VSV-G的）替换HIV-1病毒包膜。 VSV-G的扩展取向的载体，并允许通过超速13的浓度。所有的基因蛋白质编码的附件，包括VIF，VPR，VPU，和NEF被排除在包装系统。慢病毒载体的生产和操作应根据NIH的研究指引，涉及重组DNA（ <a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a> ）开展。使用慢病毒载体之前，可能需要从个别机构的生物和化学安全委员会批准。慢病毒载体转染293T细胞与慢病毒转移质粒和辅助质粒载体包装所需的蛋白质编码产生的。许多慢病毒转移质粒和辅助质粒可以得到来自Addgene，非非营利质粒库（ <a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/~~V</a> ）。一些稳定的包装细胞系已发达，但这些系统提供了灵活性和他们的包装效率普遍下降14随着时间的推移，15。市售的转染试剂盒，可支持转16的高效率，但也可以是非常昂贵的大型载体制剂。磷酸钙沉淀法转染293T细胞，并提供高效，从而提供一个可靠的和慢病毒载体生产成本的有效途径。 在这个协议中，我们生产的由磷酸钙沉淀的原则，其次是超速20％的蔗糖垫与纯化，并通过浓度，根据质粒转染293T细胞的慢病毒载体。载体滴度测定荧光激活细胞分选（FACS）肛门ysis或通过实时定量PCR。在这个协议的慢病毒载体的生产和滴定法9天就可以完成。我们提供的这些载体转导到含有神经元和神经胶质细胞的小鼠皮层文化的例子。我们表明，慢病毒载体转导和细胞类型特定的中枢神经系统的原代培养细胞中的基因表达支持高效率。

Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

在这个协议中，我们描述了慢病毒载体的生产，提纯和滴定。我们提供了一个在原代培养的神经元和神经胶质细胞的慢病毒载体介导的基因传递的例子。我们的方法也适用于其他类型的细胞<em&gt;在体外</em&gt;和<em&gt;在体内</em&gt;。

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

production-lentiviral-vectors-for-transducing-cells-from-central

Research

JoVE Journal

Neuroscience

35.8K 次观看.  Washington University School of Medicine. 在这个协议中，我们描述了慢病毒载体的生产，提纯和滴定。我们提供了一个在原代培养的神经元和神经胶质细胞的慢病毒载体介导的基因传递的例子。我们的方法也适用于其他类型的细胞在体外和在体内。

视频: Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...